Many active polypeptide therapeutics exist that have greater stability or are functional only in the C-terminal amide form, and not in the acid form. While short polypeptide amides can be conveniently made by solid phase synthesis there is still a great need for manufacturing processes able to produce longer peptides and polypeptides using cost-effective recombinant procedures coupled with post-translational amidation.
Chemical or enzymatic methods can be utilized for post-translational amide formation. Methods for amidation include the following: (a) chemical cleavage and amidation with palladium or cyanylation reagents, (b) enzymatic amidation with peptidylglycine α-amidating monooxygenase (PAM), and (c) C-terminal transpeptidation with carboxypeptidase Y. However, each of these methods carries significant disadvantages. The first method carries the disadvantage of requiring specific sequences to be incorporated into the C-terminus of the peptide, and additionally offers yields in the range of only 8-30%. The second method can effectively convert a C-terminal glycine extended peptide to an amide in some instances, but carries the disadvantage that the preparation of the required PAM enzyme is time consuming and expensive. Moreover, not all polypeptides are soluble at a pH at which the enzymatic reaction occurs. The C-terminal transpeptidation reaction is limited to the use of only a few amino acids as nucleophiles, and yields for this method can be very low and are dependent on the amino acid located on the C-terminus.
Thus, there is a need for a general and easy method of performing C-terminal amidation on recombinantly expressed polypeptides.